Detection device for laser scanning microscope

Abstract

The present invention relates to a detection device (2) for a laser scanning microscope, wherein the detection device (2) has a light input end (4), at least one filter module (14) and at least one spatially resolved detector (22) and is configured to guide light from the light input end (4) to the filter module (14) and from there to the spatially resolved detector (22), wherein the at least one filter module (14) is configured as a series of filter modules having two continuously adjustable filter elements (16), and at least one compensator element (26) is optically arranged behind the series of filter modules (14), by means of which the focal position of the light on the spatially resolved detector (22) can be adjusted.

Description

Technical Field

[0001] The present invention relates to a detection device for a laser scanning microscope, wherein the detection device has a light input end, at least one filter module and at least one spatially resolved detector and is configured to guide light from the light input end to the filter module and from there to the spatially resolved detector. Background Technology

[0002] Laser scanning microscopy is known from the prior art. A particular form of laser scanning microscopy is used, for example, in so-called "image scanning microscopy." Here, a method of fluorescence microscopy is involved, in which fluorophores, for example, arranged in a biological sample, are excited to emit light by means of focused excitation radiation, particularly by a laser beam. Here, the focused excitation beam is guided on the sample in a grid pattern, and thus excites existing fluorophores to emit light only in spatially narrowly defined regions. For each grid point, fluorescence emission is detected using a confocal detector, which, unlike the detectors used in conventional laser scanning microscopy, is spatially resolved. Therefore, for each grid point, a partial image of the sample is detected by a corresponding microscope, wherein the overall image can be calculated from the whole of these partial images. The achievable resolution of the overall image depends particularly on the quality of the imaging of the individual partial images onto the spatially resolved detector, and is higher than the resolution of conventionally confocal recorded images. Hereinafter, “image scanning microscopy” is understood to refer to every form of laser scanning microscopy with spatially resolved detection of fluorescence emission at each grid point, such as STED microscopy (stimulated emission depletion) in the case of a spatially resolved detector.

[0003] In laser scanning microscopy, various filter assemblies are used to avoid detecting the excitation light or excitation radiation, which could, for example, enter the microscope's detector as scattered light, when detecting fluorescence emission. Therefore, filter assemblies that filter out radiation at the excitation wavelength have traditionally been installed in laser scanning microscopes. However, a laser scanning microscope preferably needs to detect the fluorescence radiation of different fluorophores so that a separate microscope is not required for each fluorophore used. However, different fluorophores emit fluorescence radiation at different wavelengths, making it advantageous to use different filters in the microscope to respond to variations caused by the fluorophores used for different samples.

[0004] DE10213187 A1 describes an implementation in which multiple spectral bands or channels can be detected simultaneously by transmitting light reflected from a movable first aperture to another movable aperture. Here, a portion of the light passes through each aperture and is transmitted to a detector, which is not a spatially resolved detector. A similar device is known from DE102014116782 A1, in which light passing through an aperture plate is also spectrally separated by a prism and filtered by a movable aperture and a lens. The movable aperture, lens, and additional prism are also formed as a continuous filter module in the sense of the invention to refocus light within a defined wavelength range. However, the described detector is not spatially resolved.

[0005] In DE19835070 B4, a variable bandpass filter is used instead of a prism. This variable bandpass filter comprises a variable short-pass filter and a variable long-pass filter. The short-pass filter allows wavelengths shorter than the threshold wavelength to pass through, while the long-pass filter allows wavelengths longer than the threshold wavelength to pass through. By cleverly combining the threshold wavelengths of the short-pass and long-pass filters, it is possible to filter out a desired wavelength range. Different short-pass and / or long-pass filters can be constructed as separate filters, for example, in a filter wheel, where the wheel can rotate to move another filter into the beam path; or they can be constructed as a continuous gradient color filter (Farbverlauf filter) that can move along the longitudinal direction. However, in this configuration, the light to be detected is confocally detected by a point detector and is guided through the aperture plate before reaching the filter. In this configuration, spatially resolved detection of fluorescence emission to a grid point is also impossible. Similar devices are known from DE102006034908 A1, DE102009012874 B4, and DE102018126232 B3. In all of these documents, a color gradient filter is used as a filter element to spectrally filter light that has previously passed through an aperture plate, which is then delivered to a non-spatial-resolved detector. Summary of the Invention

[0006] Therefore, the present invention is based on the task of further developing a detection device that enables continuously adjustable spectral filtering while performing spatially resolved detection.

[0007] The present invention solves the proposed task by means of a detection device, characterized in that at least one filter module is constructed as a continuous filter module having two continuously adjustable filter elements, and at least one compensator element is optically arranged behind the continuous filter module, by means of which the focal position of light on the spatially resolved detector can be adjusted.

[0008] In existing techniques with confocal, non-spatial-resolved detectors, the size and positioning accuracy of the confocal aperture plate define the spatial resolution of the image of the sample that can be recorded. Elements located behind the aperture plate and in front of the detector do not affect the achievable resolution. Using a spatially resolved detector, rather than one integrated onto the entire detector surface, to improve the overall achievable resolution of the sample image fundamentally changes the boundary conditions and the requirements for the position, type, and manner of the optical elements used, which can be arranged in the beam path of light. If a spatially resolved detector is to be used advantageously, interference with the spatial distribution of light and spatial resolution is prohibited.

[0009] Therefore, in the prior art, it can be considered that the above-described arrangement (where spectral separation occurs after the aperture plate in the case of confocal and non-spatial-resolved detection) cannot be used in image scanning microscopy. In conventional laser scanning microscopy, the lateral and axial spatial resolution of confocal detection is essentially determined by the aperture plate. Elements located at the rear in the beam path generally do not affect the spatial resolution in this type of microscopy. However, this is different in image scanning microscopy where fluorescence emission is detected spatially at each grid point, because in this image scanning microscopy, all optical elements in front of the spatially resolved detector affect the spatial resolution of the overall image. Furthermore, it can be considered that color gradient filters, especially as continuously adjustable filter elements, have an impact on spatial resolution because they cause spatial inhomogeneities with respect to wavelength in the light passing through the filter. Furthermore, mechanical movement of individual optical components within the beam path (e.g., necessary for creating the detection band when using color gradient filters or prism assemblies) causes changes in the optical path and thus alters the imaging characteristics of the detection device in both diffraction-limited and desired spatially resolved detectors. Lateral shift occurs, particularly at the focal point on spatially resolved detectors.

[0010] This invention is based on the understanding that cascading filters along the path of light to a spatially resolved detector has minimal impact on the spatial resolution. This allows the use of color gradient filters. The effects of mechanical movement of the various components in the light beam path can be compensated for by a compensator element, which allows adjustment of the incident position of the light on the spatially resolved detector.

[0011] According to the invention, the detection device has at least one filter module configured as a continuous filter module. It has two continuously adjustable filter elements. These filter elements are preferably adjustable independently of each other. Thus, the component of the incident light fed to the at least one spatially resolved detector is the spectral component of the incident light, which has an upper wavelength and a lower wavelength. The upper wavelength is preferably determined by one of the filter elements, and the lower wavelength is determined by the other. If the two filter elements can be adjusted independently of each other, the difference between the upper and lower wavelengths can also be adjusted, and thus the spectral width of the light component guided to the spatially resolved detector can be adjusted. If the two filter elements can only be adjusted together, for example, in the case of an aperture plate in the beam path of a spectrally separated light beam by a prism, the spectral width of the light reaching the detector cannot be adjusted. Such embodiments are also covered by the selected description.

[0012] At least one of the filter modules is formed by two continuously adjustable filter elements. A “continuously adjustable filter element” is understood to be a filter element that can be used as a short-pass filter or a long-pass filter, and whose limiting wavelength can be continuously, i.e., stufenlosively, adjusted. Thus, a continuously adjustable short-pass filter allows light with wavelengths shorter than the limiting wavelength to pass through. Here, the limiting wavelength can be freely adjusted. Similarly, a continuously adjustable short-pass filter allows light with wavelengths longer than the limiting wavelength to pass through, where the limiting wavelength can also be freely adjusted. By combining the short-pass and long-pass filters, light within a defined wavelength range can be filtered out from a wider spectral range. This light is then fed to a detector, preferably the at least one spatially resolved detector.

[0013] A continuous filter module with two continuously adjustable filter elements can be used as a transmission filter or a reflection filter. In the case of a transmission filter, a portion of the light that passes through the two continuously adjustable filter elements in a transmitted form is fed to the detector, while in the case of a reflection filter, a portion of the light that is reflected at the filter elements is fed to the detector. Using a transmission filter is advantageous.

[0014] Moving at least one of these two continuously adjustable filter elements typically requires mechanical movement of the corresponding filter element. This can be motor-driven, for example by an electric motor, or achieved using a piezoelectric element for higher precision.

[0015] Regardless of how the movement of the filter element is caused, the filter element must be movably supported. This is due to manufacturing tolerances, errors, and gaps, which can cause the orientation of the filter element relative to the optical path to change. This can lead to a shift in the filtered light, that is, a shift in the focal point of the filtered light above the spatially resolved detector. This can be compensated for by a compensator element.

[0016] According to the invention, the at least one compensator element is optically arranged behind a series of filter modules. This means that the compensator element does not affect the light in the path leading to the filter modules. Therefore, the compensator element can only move or otherwise affect a component arranged behind the filter modules in the beam path. Alternatively or additionally, the compensator element can also be used to affect the light that has left the filter modules.

[0017] Preferably, the at least one filter module is part of a filter cascade having at least two filter modules. This makes it possible to detect multiple spectral channels, i.e., wavelength ranges, of light incident on the detection device through the light input without altering the settings on the filter cascade or the detection device. The number of channels detectable in this way corresponds to the number of filter modules in the filter cascade or the number of filter modules in the filter cascade plus one. Two channels can be detected using a single filter module.

[0018] In a preferred configuration, the at least one compensator element is configured to move the at least one spatially resolved detector. This preferably includes movement in a plane perpendicular to the optical axis, i.e., lateral movement. Alternatively or additionally, the movement may also include movement of the detector along the optical axis, i.e., axial movement.

[0019] Preferably, at least one compensator element is arranged between the consecutive filter modules and the spatially resolved detector. Here, "between" does not necessarily mean located between the two in terms of location or geometry, even though this is the preferred configuration. The only important thing is that the at least one compensator element is optically arranged between the filter modules and the detector, i.e., configured to influence the light along the path from the filter modules to the detector.

[0020] Preferably, at least one compensator element, particularly preferably each compensator element, is constructed as a movable, especially tiltable, mirror, which is further preferably tiltably supported about two tilting axes. Particularly preferably, the two tilting axes are perpendicular to each other. Typically, it is sufficient to adjust the focal position of the light on the spatially resolved detector (i.e., the orientation of the point conjugate to the center of the focal point in the sample) in two directions extending along the detector plane (i.e., perpendicular to the optical axis and the optical path). Therefore, these two preferably perpendicular directions (referred to as the X and Y directions) extend parallel to the detector plane. In a preferred configuration, it is also possible, through the at least one compensator element, to change the focus of the incident light on the spatially resolved detector, i.e., to achieve a movement of the focal position in a vertical third direction (Z direction). This is preferably achieved by moving a lens in the beam path. Such lenses are known, for example, to focus light guided from the filter cascade to the spatially resolved detector onto the detector surface. In a preferred embodiment, the lens (which may also be a lens arrangement having multiple optical components, especially multiple lenses) is movably arranged along an optical axis, preferably corresponding to the optical path. In this case, the lens is also a compensator element, or it is also a compensator element, because it allows adjustment of the focal position of the light on the spatially resolved detector. Although mechanical movement of the individual optical components themselves does not cause a movement of the focal position in the Z direction, or at most a very small movement, the focal position in the Z direction is associated with the selection of the detection band due to chromatic aberration in the imaging characteristics. In conventional laser scanning microscopes, as is known from the prior art, the aperture plate used here is usually adjusted to be good on average for all detection bands to be used, but at least not perfect for all bands. Using a spatially resolved detector eliminates the need for an aperture plate. Combined with a compensator element for adjusting the focal position of the light on the spatially resolved detector in the Z direction, additional adjustment possibilities for improved imaging quality are thus provided. The compensator element can have multiple different elements (e.g., tiltable mirrors and movable lenses), which are preferably not necessarily used to adjust the focal position of light on the detector in different directions. Multiple compensator elements acting on light from the same filter module are called a compensation device. Different compensator elements of a compensation device can affect the focal position in different directions. It is advantageous that each compensator element affects the focal position in only one spatial direction. However, it is also possible that individual or all compensator elements of the compensation device affect the focal position in multiple spatial directions.

[0021] For some applications, it is advantageous to position the compensator element between the filter module and the spatially resolved detector. Positioning it between the two components means that the compensator element is located within the beam path from the filter module to the spatially resolved detector. The compensator element does not necessarily need to be structurally located between the filter module and the detector, as long as it is situated within the beam path between the two components.

[0022] Preferably, the continuously adjustable filter elements are color gradient filters, which are arranged to be movable in the longitudinal direction. They function as short-pass and long-pass filters, with different color gradient filters used for different filter types. Both color gradient filters are movable along the longitudinal direction, which corresponds to the direction in which the filter's gradient extends. The limiting wavelength of such a color gradient filter depends on the position of the light incident on it. Therefore, the limiting wavelength changes, thus creating a gradient. The color gradient filters are arranged to be movable along this direction.

[0023] In a preferred configuration, the detection device has multiple detectors. Here, the number of detectors advantageously corresponds to the number of filter modules, which limit the number of spectral channels detectable without changes to the microscope structure. Therefore, it is advantageous that the number of detectors is the same as the number of filter modules. Preferably, the multiple detectors are configured as spatially resolved detectors. Particularly preferred is that all detectors used are spatially resolved detectors.

[0024] Advantageously, a compensator element is provided in front of each spatially resolved detector, by means of which the incident position of light on the corresponding spatially resolved detector can be adjusted. The compensator elements for different spatially resolved detectors can be constructed identically or differently from each other. Preferably, they can be controlled independently of each other, such that the incident position of light on different spatially resolved detectors can be separated and adjusted independently. In a filter cascade, light fed to the respective detector passes through different numbers of filter elements. Thus, after the light has passed through only the first filter module, a portion of the light is fed to the first detector. Another portion of the light is fed to the second detector and has already passed through two filter modules. The more mechanically adjustable filter elements included in these different filter modules, and which can be adjusted, the greater the correction of the incident position of light on the spatially resolved detector, which is necessary to achieve and maintain the desired spatial resolution and quality of imaging. Therefore, the compensator elements are preferably operable and adjusted separately, independently, and / or individually.

[0025] Preferably, each filter module has a detector output through which light is guided to at least one detector. Furthermore, each filter module, except for the last one, has a filter output through which light is guided to another filter module. The last filter module can also have a filter output, after which another filter module can be arranged. This filter output of the last filter module can serve as another detector output, or it can lead to a beam trap. It has proven advantageous to arrange a detector after each detector output to capture and detect light exiting the corresponding filter module through the detector output and transmit it to the analysis and processing apparatus.

[0026] In one embodiment, the detection device has at least one switching element that can guide light from at least one filter module to different detectors. The switching element is, for example, constructed as a tilting mirror. The switching element is configured to guide light from the filter module, preferably exiting through the detector output and being guided onto the switching element, to different detectors. For this purpose, the switching element can be placed in different states or conditions in which light from the filter module is guided to different detectors when it strikes the switching element.

[0027] Alternatively or additionally, a switching element can be used to direct light from multiple filter modules to a detector, preferably a spatially resolved detector. This reduces the number of detectors required, especially spatially resolved detectors, thus saving costs. In this configuration, the switching element can be constructed as a tilting mirror. Depending on the state of the switching element, i.e., especially the tilting mirror, light from different filter modules is directed to the desired detector. Preferably, with a single switching element, light from only a single filter module is directed to the detector. Rapid switching can be achieved using modern switching elements, especially tilting mirrors.

[0028] Preferably, the detector arrangement has multiple switching elements, preferably as many as the number of filter modules. Each switching element is then configured to direct light from at least one filter module to a different detector. If a separate switching element exists for each filter module, light leaving the filter module can be directed to the desired detector, which can be selected independently of the detector chosen for light from another filter module.

[0029] Instead of being provided here, at least one switching element is provided, which redirects light from multiple filter modules. For this purpose, light from the respective filter module is guided to the switching element via an optical element (e.g., a mirror) and from there distributed to the corresponding detector. This reduces the number of switching elements required. In this configuration, the detector selected from the multiple filter modules for light is not independent of each other.

[0030] The multiple or single switching elements enable the rapid, sequential acquisition of high-resolution image recordings of samples in different color channels, for example, when using different fluorophores, wherein a spatially resolved detector is used for each color channel, although not every single color channel or filter module has a spatially resolved detector assigned only to that module. This reduces the number of spatially resolved detectors required. This is particularly possible with configurations of switching elements that allow light from different filter modules to be directed to a single spatially resolved detector. Thus, preferably, it is sufficient if the detection device has only a single spatially resolved detector.

[0031] Image recording can then be performed, for example, line by line, wherein, in the first state of the switching element, the first color channel is recorded along the line using the first filter module, wherein the switching element is switched when the scanning unit returns to the beginning of the line, and wherein the second color channel is recorded along the line using the second filter module. This process can continue until image lines have been recorded for all desired color channels, but at most for each existing filter module. Alternatively, it is also possible to switch the switching element at the end of each line and record the next line separately in opposite scanning directions. Subsequently, this process is repeated, for example, in the same order, or alternatively, in a different order of the color channels used for subsequent image lines. It is also possible to use a recording scheme in which the scanning unit scans a new line each time it passes through, such that the individual color channels are recorded interleaved. Such a recording scheme is common in laser scanning microscopy, where the line spacing is chosen such that the spacing between lines of a single color channel matches the desired resolution of the overall image. The switching element (e.g., a tilting mirror with a previously determined tilt state) can switch to its determined switching state with such high reproducibility that the adjustment of the compensator element assigned to the filter module must only be performed once, after the filter module is adjusted to select the probe band, when recording for multiple color channels begins.

[0032] Preferably, the switching element is constructed as a compensator element. In this case, after adjusting the filter module for the probe band of each color channel, the switching state of the integrated switching element and the compensator element is determined such that the error allocated to the corresponding filter module is compensated after each switch to one of the determined switching states.

[0033] Preferably, the detection device has an optical arrangement such that the waist of the incident Gaussian beam lies between two adjacent filter modules or within one filter module. This preferably reduces beam divergence. Adjacent filter modules are preferably arranged at the center of the filter cascade or at least in its central region. If the filter cascade has, for example, four filter modules, the waist is preferably located between the second and third filter modules. If the filter cascade has five filter modules, the waist is preferably located within the third filter module or between the third and fourth filter modules. In this way, the divergence that is inherent in a Gaussian beam with a small waist has as little impact as possible on the filtering of the incident light through the filter cascade. The smaller the waist of the Gaussian beam, the greater the divergence. When using continuously adjustable filter elements, especially color gradient filters, it is important that the corresponding light spot formed by the incident light on the respective filter element is as small as possible, so as to keep the spatial correlation of the limiting wavelength inherent in the color gradient filter within the light spot as small as possible. An excessively large beam diameter can also cause the mirrors that transmit the beam within the cascade to be unable to transmit the entire beam, and thus the beam is truncated. If the waist of the Gaussian beam is placed, for example, in front of the first filter module, the Gaussian beam will continuously widen as it passes through the filter cascade, making the light spot, especially on the last filter module and its filter elements, very large. Therefore, this arrangement is avoided. The optimal value of the beam waist diameter depends on the precise structural configuration of each filter module and the number of modules. In the specific arrangement of the invention, the detection device operates by means of a Gaussian beam with a waist having a diameter of less than 1.5 mm, preferably less than 1 mm.

[0034] This is achieved by positioning the waist in the middle region of the filter cascade. The light spot on the first filter module and its filter elements initially decreases as the light advances along the optical path until it reaches the waist. Conversely, after the waist, the light spot widens and becomes larger. However, overall, the size of the light spot on all continuously adjustable filter elements remains within an acceptable range.

[0035] Preferably, the detection device has an electrical or electronic controller configured to control the compensator element such that the maximum value of the radiation detected by the spatially resolved detector is at the center of the spatially resolved detector. The electronic controller can, for example, be part of an electronic data processing device, which may be part of a microscope or a detector assembly. Since the spatially resolved detector already measures the distribution of incident light intensity, adjustment parameters can be determined from the detected signal to control the compensator element.

[0036] In a preferred configuration, a spatially resolved detector—and particularly preferably all spatially resolved detectors—is a segmented planar detector. This includes pixel detectors having a plurality of individual detectors spatially separated from each other, such as SPAD (single-photon avalanche diode) arrays. At least one lens or lens arrangement is preferably arranged in front of them, configured to image incident light onto the detector. Here, the lens or lens arrangement is preferably arranged such that a first maximum, a first minimum, and a second maximum value of the diffraction spot are detected in a spatially resolved manner. Alternatively, the first maximum and the first minimum value can also be detected.

[0037] The at least one compensator element according to the invention functions as part of a laser scanning microscope during the operation of the detection device to compensate for the movement of filtered light (i.e., the focal position of the filtered light on the spatially resolved detector) caused by the movement of at least one of the filter elements. This should occur as quickly as possible to avoid unnecessarily delaying measurements. The laser scanning microscope has at least one light source and optics that focus the light from the light source onto the focal region of the sample. This illumination light is an excitation light used to excite a fluorophore located in or on the sample. The illumination light is scanned or rasterized on the sample by a scanning unit. The fluorophore then emits emission light that should be detected. This emission light is delivered to the detection device via the scanning unit and filtered there by the at least one filter module, and then fed to the at least one spatially resolved detector. Here, the detector (more precisely, the detector aperture) is arranged confocally on or in the sample with the focal region. This confocality can be disturbed by the movement of at least one filter element and then restored by the compensator element.

[0038] Therefore, it is necessary to determine how the compensator elements used to compensate for interference should be adjusted. An apparatus and method for readily obtaining this information are described in an unpublished international patent application, PCT / EP2020 / 066589. The entire contents of that application are incorporated herein by reference.

[0039] This determination can be achieved, for example, in multiple steps using a device for checking confocality. Light emitted from the light source is fed to an auxiliary detector via a scanning unit, not to the sample. This auxiliary detector (more precisely, its auxiliary detector aperture) is positioned in the focal plane and scanned by the illumination light by adjusting the scanning unit. Here, the first intensity distribution of the illumination light detected by the auxiliary detector is detected.

[0040] In another step, auxiliary light is introduced into the scanning unit by means of an auxiliary light source—preferably also arranged in the same focal plane as the auxiliary detector and on the optical axis associated with the auxiliary detector aperture—which is then fed to the detection device by the scanning unit. Here, the auxiliary detector aperture and the auxiliary light source can be formed from the same element, such as a diode or the end of an optical fiber, which can function as both a transmitter and receiver, and the optical fiber is connected via a fiber coupler to not only the detector but also the light-emitting device (e.g., a laser). Here, the detection device (more precisely, its detector aperture) is also scanned by adjusting the scanning unit, and thus a second intensity distribution is detected. Then, at least one difference between the first and second intensity distributions across different states of the scanning unit is detected as a measure of the confocality error. This error is determined, and adjustment parameters for the compensator element are derived from it, and the compensator element is adjusted so that the error is compensated. Hereinafter, the combination of the auxiliary light source and the auxiliary detector aperture is referred to as the auxiliary device. This applies even if the auxiliary detector aperture and the auxiliary light source are formed from the same component, such as a diode.

[0041] Through these two steps, the deviation in confocality can be determined particularly simply and quickly, and the compensator element can be appropriately adjusted. Since the detection device according to embodiments of the invention can be configured to detect different spectral ranges, i.e., different colors, it is advantageous that the laser scanning microscope has multiple auxiliary devices, each with an auxiliary light source. Different auxiliary light sources emit or are at least capable of emitting auxiliary light with wavelengths different from each other. Furthermore, each auxiliary device has an auxiliary detector aperture configured to detect the light from the auxiliary light source of the corresponding auxiliary device. Therefore, the auxiliary detector apertures of different auxiliary devices can detect light with wavelengths different from each other. Thus, multiple auxiliary devices can be arranged in a simple manner, because the exact position of such a pair within the focal plane is not important; what matters is that the pair is arranged within the focal plane of the scanning area of ​​the scanning unit. If multiple auxiliary devices are present, the above method can also be performed simultaneously for multiple spectral components (i.e., multiple detection channels). Each auxiliary device with an auxiliary light source and an auxiliary detector aperture preferably operates normally at a predetermined wavelength, the auxiliary light source emitting auxiliary light, and the auxiliary detector aperture detecting the light emitted from the auxiliary light source. The auxiliary light source and the auxiliary detector aperture are coordinated with each other regarding this wavelength, which can also be referred to as the operating wavelength of the auxiliary device. If multiple auxiliary devices are used, these multiple auxiliary devices preferably have different operating wavelengths.

[0042] The present invention also addresses the proposed task using a laser scanning microscope having the detection apparatus described herein. Attached Figure Description

[0043] Hereinafter, some embodiments of the invention will be described in more detail with the aid of the accompanying drawings. The drawings show:

[0044] Figure 1 A schematic diagram of a detection device according to a first embodiment of the present invention is shown;

[0045] Figure 2 A partial view of a detection device according to another embodiment of the present invention is shown;

[0046] Figure 3 Shown from Figure 2 Partial views in different settings;

[0047] Figure 4 A partial view of a detection device according to another embodiment of the present invention is shown;

[0048] Figure 5 and Figure 6 A schematic diagram showing the measured intensity. Detailed Implementation

[0049] Figure 1 A detection device 2 according to a first embodiment of the invention is schematically shown. It has a light input 4 through which light previously guided through other optical devices 6 (of which only illustrative examples are shown) enters the detection device 2. A telescope 8 is schematically shown. Through this telescope, the waist of the incident Gaussian beam, in the illustrated embodiment, shifts to the right between two first filter modules 14. The detection device 2 has a filter cascade 12, which, in the illustrated embodiment, has three filter modules 14. Each of the filter modules 14 has two filter elements 16, which are continuously adjustable and can be configured, for example, as color gradient filters. The two illustrated first filter modules 14 each have a detector output 18 and a filter output 20. The portion of the light beam 10 that passes through the two filter elements 16 in the first filter modules 14 is guided from the detector output 18 to a spatially resolved detector 22. The portion of the light beam 10 reflected at the first filter element in the filter element 16 within the first filter module 14 is guided from the filter output 20 to the next filter module 14. The last filter module 14 (which...) Figure 1 (Shown on the far right) has two detector outputs 18, because the light emitted from each output is not sent to another filter module 14, but is instead sent to the detector and beam trap 25 configured as a spatially resolved detector 22. Figure 1 Compensator element 26, not shown, is optically located behind filter element 16 of filter module 14. This compensator element can be assigned to corresponding detectors 22, 24 and can be used to change the focal position of light on detectors 22, 24. Compensator element 26 can, for example, be used to move imaging lens 32 perpendicular to the beam direction, wherein this movement (shown by movement arrow 27' in the figures) adjusts the focal position in the Z direction. Other compensator elements cause movement in the X and Y directions (shown by movement arrow 27 in the figures). For clarity, in Figure 1 In this configuration, the movement arrows 27 and 27' are assigned to only two of the three filter modules 14. The corresponding, not shown, compensator element 26 is also assigned to the third filter module. This allows for sufficient compensation.

[0050] Figure 2 A schematic partial view of the detection device 2 is shown, and three filter modules 14, each with two filter elements 16, are presented. (By...) Figure 2The detector output terminal 18, pointing downwards, receives a portion of the light that exits the corresponding filter module 14 and is guided onto mirror 28. This mirror 28 reflects this portion of the light onto compensator element 26, which, in the illustrated embodiment, is configured as a movable mirror. It also functions as switching element 36.

[0051] In the presented configuration, switching element 36 redirects light that has exited the first filter module 14 via its detector output 18 to the spatially resolved detector 22, and redirects light that has exited the detector outputs 18 of the second and third filter modules 14 to the non-spatially resolved collection detector 24.

[0052] exist Figure 3 In the configuration presented, the switching element 36 guides the light that has left the second filter module 14 through its detector output terminal 18 to the spatially resolved detector 22, and guides the light that has left the first filter module 14 through its detector output terminal 18 to the beam trap 25. At the same time, it guides the light that has left the third filter module 14 through its detector output terminal 18 to the collecting detector 24, which is adjacent to the spatially resolved detector 22.

[0053] In the unpresented third configuration, it guides the light that has exited the third filter module 14 through its detector output 18 to the spatially resolved detector 22, while guiding the remaining light to the beam traps 25 respectively. Here, the switching element 36 also functions as the compensator element 26. This means that the settings determined by the switching element 36 are such that when the selected detection band changes (i.e., when the settings of the filter modules 14 assigned to the spatially resolved detectors 24 change), the error in the focal position of the light on the spatially resolved detector 22 is compensated. Matching the position (Lage) of the light on the collecting detectors is not possible, but also unnecessary, because their detection apertures are chosen to be large enough. Therefore, in the configuration shown in this embodiment, the compensator element 26 is arranged such that the light emitted from the detector outputs 18 of the second and third filter modules is guided to the two collecting detectors 24, while the light emitted from the detector outputs 18 of the first filter module 14 is directed to the spatially resolved detector 22. The compensator element 26, configured as a movable mirror, also serves as a switching element 36, which guides light emitted from the detector output of one of the filter modules 14 to the spatially resolved detector 22. In an alternative configuration, the mirrors 28 can also be part of a compensation device having two compensator elements 26 configured as two mirrors. The respective mirrors 28 can then be used, for example, to achieve compensation in the first direction. In this case, the second mirror (i.e., preferably the switching element 36) is used to achieve compensation in a second direction, which is preferably perpendicular to the first direction.

[0054] Figure 3 Showing from Figure 2 A known structure includes a filter module 14 and its detector output terminal 18. The emitted light is guided onto a mirror 28, which then directs the light onto a compensator element 26. This is in... Figure 3 Presented in Figure 2 In different locations within the array, the light from the first filter module is directed into the beam trap 25 and is not available for analysis. The light is absorbed in the beam trap 25 and therefore cannot fall as scattered light onto one of the detectors 22 and 24. The light from the middle filter module 14 is directed onto the spatially resolved detector 22, and the light from the right-hand filter module 14 is directed onto the collecting detector 24. Since the incident position of the light on the respective detectors 22 and 24 is only important for the spatially resolved detector 22, a single compensator element 26 is sufficient in the illustrated embodiment, which has only a single spatially resolved detector 22.

[0055] Figure 4Another configuration is shown. This configuration also has three filter modules 14, each with two filter elements 16, which are configured to be continuously adjustable. These filter modules 14 also each have a detector output 18, from which light... Figure 4 The light exits the corresponding filter module 14 downwards through the detector output terminal in a direction toward the corresponding spatially resolved detector 22. The light emitted through a detector output terminal 18 is respectively incident on the compensator element 26, and from there is guided by the corresponding imaging lens 32 to the corresponding spatially resolved detector 22. The switching element 36 is not necessary. Figure 5 and Figure 6 Exemplary measurements are shown. Each field 34 corresponds to one pixel of the spatially resolved detector 22. During sample scanning, a large number of partial images are recorded using the spatially resolved detector 22, in which each pixel, i.e., each field 34, detects incident photons, i.e., specifically the intensity of the incident light. Figure 5 and Figure 6 The values ​​assigned to each field 34, as shown, correspond to the summation of intensities across multiple recorded partial images. Therefore, this is a measure of the intensity detected by the respective field 34, integrated across multiple images. Here, these values ​​have been normalized so that the maximum value is 1.00. If a very large number of photons are detected and a sufficiently large area of ​​the sample is scanned, a rotationally symmetric distribution will be produced on average in an optimally tuned apparatus. Such a distribution does not occur in… Figure 5 As shown in the figure, the maximum value shifts to the lower left. Figure 6 Although the maximum value is in the central field 34, the distribution of values ​​around it is approximately rotationally symmetric. Deviations in rotational symmetry are particularly likely due to photon noise, insufficient scanning areas of the non-uniform sample, and incomplete compensation. In principle, however, the compensation device and its compensator element 26 can be adjusted based on the summed intensity to compensate for errors in the focal point position. If the sample is loaded with both excitation light and STED light during scanning, which suppresses fluorescence emission into the region outside the excitation focal point, the determination of compensation can be simplified. This has the effect that fluorescence is emitted only from the region near the axis of the excitation focal point, making each individually detected intensity distribution more approximately rotationally symmetric. Sample non-uniformity is less important. In this way, the apparatus, especially the microscope, can be adjusted and configured without a reference sample or special devices for adjusting confocality.

[0056] List of reference numerals

[0057] 2. Detection device

[0058] 4 Optical Input Terminal

[0059] 6. Optical devices

[0060] 8 Telescopes

[0061] 10 beams of light

[0062] 12 Cascaded Filters

[0063] 14 Filter Module

[0064] 16 Filter Elements

[0065] 18. Detector output terminal

[0066] 20 Filter Output

[0067] 22 Spatial-resolution detectors

[0068] 24. Collect detectors

[0069] 25 beam traps

[0070] 26 Compensator Components

[0071] 27, 27' Moving arrows

[0072] 28 mirrors

[0073] 30 Absorption Elements

[0074] 32 Imaging Lenses

[0075] 34 games

[0076] 36 Switching elements

Claims

1. A probe device (2) for a laser scanning microscope, wherein The detection device (2) has - a light input (4), - at least one filter module (14), and - at least one spatially resolving detector (22), which detects the diffraction spot in a spatially resolving manner, the detection device being designed to guide the light from the light input (4) to the filter module (14) and from there to the spatially resolving detector (22), characterized in that at least one filter module (14) is designed as a continuous filter module with two continuously adjustable filter elements (16), at least one compensator element (26) being arranged optically behind the continuous filter module, by means of which the focal position of the light on the spatially resolving detector (22) can be set, in that the at least one filter module (14) is part of a filter cascade (12) with at least two filter modules (14), wherein, with the exception of the last filter module (14), each filter module (14) has a detector output (18) by means of which the light is guided onto a detector and a filter output (20) by means of which the light is guided onto another filter module (14).

2. The probe device (2) according to claim 1, characterized in that The detection device (2) has an optical device, the detection device being designed in such a way that the waist of an incident Gaussian beam lies between two adjacent filter modules (14) or within one filter module (14).

3. The probe device (2) according to claim 2, characterized in that The optical device is a telescope (8).

4. The probe device (2) according to any one of claims 1 to 3, characterized in that At least one compensator element (26) is designed to move the spatially resolving detector.

5. The probe device (2) according to claim 1, characterized in that At least one compensator element (26) is arranged between the continuous filter module and the spatially resolving detector.

6. The probe device (2) according to any one of claims 2 to 3, characterized in that At least one compensator element (26) is arranged between the continuous filter module and the spatially resolving detector.

7. The probe device (2) according to claim 6, characterized in that At least one compensator element (26) is designed as a movable mirror (28).

8. The probe device (2) according to claim 7, characterized in that The movable mirror (28) is tiltable about at least two tilt axes.

9. The probe device (2) according to claim 5, characterized in that At least one compensator element (26) is designed as a movable mirror (28).

10. The probe device (2) according to claim 9, characterized in that The movable mirror (28) is tiltable about at least two tilt axes.

11. The probe device (2) according to any one of claims 1 to 3, 5, 9, 10, characterized in that The continuously adjustable filter elements (16) are color ramp filters, which are arranged to be movable in the longitudinal direction.

12. The probe device (2) according to any one of claims 1 to 3, 5, 9, 10, characterized in that The detection device (2) has a plurality of detectors.

13. The probe device (2) according to claim 12, characterized in that The detection device (2) has the same number of detectors as filter modules (14) or the same number of detectors as detector outputs (18).

14. The probe device (2) according to claim 13, characterized in that A plurality of the detectors are designed as spatially resolving detectors (22).

15. The probe device (2) according to claim 14, characterized in that All detectors are designed as spatially resolving detectors (22).

16. The probe device (2) according to claim 14, characterized in that In front of each spatially resolving detector (22) a compensator element (26) is arranged, by means of which the focal position of the light on the spatially resolving detector (22) can be set. In front of each spatially resolving detector (22) a compensator element (26) is arranged, by means of which the focal position of the light on the spatially resolving detector (22) can be set.

17. The probe device (2) according to claim 15, characterized in that In front of each spatially resolving detector (22) a compensator element (26) is arranged, by means of which the focal point position of the light on the spatially resolving detector (22) can be set.

18. The probe device (2) according to any one of claims 1 to 3, 5, 9, 10, characterized in that The detection device (2) has at least one switching element (36) by means of which the light can be guided from at least one filter module (14) to different detectors and / or by means of which the light can be guided from a plurality of filter modules (14) onto a spatially resolving detector.

19. The probe device (2) according to claim 11, characterized in that The detection device (2) has at least one switching element (36) by means of which the light can be guided from at least one filter module (14) to different detectors and / or by means of which the light can be guided from a plurality of filter modules (14) onto a spatially resolving detector.

20. The probe device (2) according to claim 18, characterized in that The at least one switching element (36) is a compensator element (26).

21. The probe device (2) according to claim 19, characterized in that The at least one switching element (36) is a compensator element (26).

22. The probe device (2) according to any one of claims 1 to 3, 5, 9, 10, characterized in that The detection device (2) has an electrical or electronic controller which is set up to control the compensator element (26) such that a maximum of the radiation detected by the spatially resolving detector (22) is in the center of the spatially resolving detector (22).

23. The probe device (2) according to claim 11, characterized in that The detection device (2) has an electrical or electronic controller which is set up to control the compensator element (26) such that a maximum of the radiation detected by the spatially resolving detector (22) is in the center of the spatially resolving detector (22).

24. Laser scanning microscope having a detection device (2) according to any one of claims 1 to 23.

25. The laser scanning microscope of claim 24, wherein, The laser scanning microscope has a device for checking the confocality, which device comprises an auxiliary device having an auxiliary light source and an auxiliary detector aperture, which are jointly arranged on a common optical axis in a focal plane.

26. The laser scanning microscope of claim 25, wherein, The device for checking the confocality has a plurality of auxiliary devices, the auxiliary light sources of which have different operating wavelengths.

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